US9623149B2 - Method for producing an acellular dermal matrix, and acellular dermal matrix produced by same - Google Patents

Method for producing an acellular dermal matrix, and acellular dermal matrix produced by same Download PDF

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US9623149B2
US9623149B2 US13/580,248 US201013580248A US9623149B2 US 9623149 B2 US9623149 B2 US 9623149B2 US 201013580248 A US201013580248 A US 201013580248A US 9623149 B2 US9623149 B2 US 9623149B2
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cryoprotectant
acellular dermal
dermal matrix
skin
buffer
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US20120329034A1 (en
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Wook Chun
Weon Ik CHOI
Joon Pio Hong
Hyun Seung Ryu
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Cgbio Co Ltd
CG Bio Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/362—Skin, e.g. dermal papillae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention relates to a method for preparing an acellular dermal matrix (ADM) and an acellular dermal matrix prepared therefrom. More specifically, the present invention relates to a method in which a cryoprotectant is prepared by adding sucrose to basic constituents comprising glycerol, propylene glycol, and a basic solvent or solution, and the resulting solution is then penetrated into skin tissue in which epidermis and cells in dermis are removed to prepare an acellular dermal matrix.
  • ADM acellular dermal matrix
  • a cryoprotectant is prepared by adding sucrose to basic constituents comprising glycerol, propylene glycol, and a basic solvent or solution, and the resulting solution is then penetrated into skin tissue in which epidermis and cells in dermis are removed to prepare an acellular dermal matrix.
  • Skin is the largest organ, covering the entire human body, and has functions of preventing loss of body fluid, influx of toxic substances and microbes from the outside, and protecting the body from physical and chemical stimuli.
  • a protective membrane is needed to prevent infection of impaired regions and the loss of body fluid, along with not leaving a scar at the impaired region and preventing serious shrinkage accompanied by the process of spontaneous cure.
  • impaired skin tissue there are three methods of autograft in which a patient's own skin is transplanted, allograft in which the skin of another human being is transplanted and xenograft in which the skin of an animal is transplanted.
  • autograft is the most ideal. However, when burnt areas are extensive, there is a limitation of the regions from which skin tissue may be obtained, and the harvesting regions can leave a new scar. Allograft plays a greater role in helping the movement of cells at the periphery of the impaired region and curing than permanent engraftment.
  • the dead epidermis and dermis layers are removed and skin grafting is then carried out by using an acellular dermis in which the epidermis and cells in the dermis are removed from skin harvested from a corpse to avoid immunorejection.
  • Cultured keratinocytes will then complete the entire skin thereon. Because such a completed skin includes basement membrane, it can play a role in protecting the body from external hazardous substances.
  • U.S. Pat. No. 5,336,616 discloses a method for preparing collagen-based tissues for transplantation.
  • tissues prepared according to the method disclosed in the above patent have problems in that cryoprotectant ingredients do not sufficiently penetrate into collagen tissues, and low concentration of sugar cannot sufficiently exclude moisture from collagen tissues—which has a characteristic of absorbing lots of moisture—so that many ice crystals are formed at the time of freezing. Such ice crystals make many pores in tissues in the course of drying and destroy collagen tissues, and the tissues are rapidly degraded after transplantation so as not to maintain their structure and to be absorbed.
  • the technical problem to be solved in the present invention is to provide a new method for preparing an acellular dermal matrix which can efficiently increase tissue stability and minimize change of biological properties compared with conventional methods.
  • the present invention provides a method for preparing an acellular dermal matrix comprising the steps of:
  • the present invention also provides an acellular dermal matrix which is prepared by the above method.
  • epidermis and cells in dermis of allograft skin are removed to avoid immunorejection.
  • the removal of epidermis and cells in dermis may be carried out according to various methods known in the art, and there is no special limitation thereto.
  • the removal of epidermis may be carried out, for example, by treatment with enzymes such as trypsin, collagenase or dispase, or NaCl solution.
  • the removal of cells in dermis may be carried out, for example, by treatment with Triton X100, Tween 20, Tween 40, Tween 60, Tween 80, SDS (sodium dodecylsulfate) and the like.
  • glycerol, propylene glycol, and a basic solvent or solution are used as basic constituents of a cryoprotectant.
  • the basic solvent or solution refers to a solvent or solution which acts as a base for the preparation of the cryoprotectant, and distilled water, normal saline, a buffer solution which is used in treating animal cells or an animal cell culture medium may be used.
  • the buffer solution which is used in the treatment of animal cells—may be used without specific limitation.
  • the example of the buffer solution includes, but is not limited to, PBS (phosphate-buffered saline), HBSS (Hank's balanced salt solution), TBS (Tris-buffered saline), TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, Bicine (N,N-Bis(2-hydroxyethyl)glycine) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer, PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2-(N-morpholino)ethanesulfonic acid) buffer and the like.
  • the animal cell culture medium used may be any medium known in the art.
  • the example of the animal cell culture medium includes, but is not limited to, MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI 1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE (bovine pituitary extract)), Keratinocyte-SFN (with BPE), KnockOut D-MEM, AminoMAX-II Complete Medium and AminoMAX-C100 Complete Medium.
  • MEM Minimum Essential Media
  • DMEM Dulbecco's Modified Eagle Media
  • RPMI 1640 RPMI 1640
  • IMDM Iscove's Modified Dulbecco's Media
  • Keratinocyte-SFM without BPE (bovine pituitary extract)
  • Keratinocyte-SFN with BPE
  • KnockOut D-MEM AminoMAX
  • glycerol, propylene glycol and the basic solvent or solution may be preferably used in a mixing ratio of 0.5 ⁇ 2:0.5 ⁇ 2:6 ⁇ 10, more preferably 0.8 ⁇ 1.5:0.8 ⁇ 1.5:7 ⁇ 9, most preferably 1:1:8, based on weight.
  • the mixing ratio of glycerol and propylene glycol is less than 0.5, there may be a problem of freezing damage in a freezing step. If the mixing ratio of glycerol and propylene glycol is greater than 2, there may be a problem of the denaturation of tissue after freeze-drying.
  • a cryoprotectant is prepared by dissolving sucrose in the solution in which the basic constituents are mixed to the final concentration of 20 to 40% by weight.
  • freezing is carried out by using a slow freezing method, loss of moisture in cells and tissues can occur. As a result, ice crystals are formed in cells, and cells and tissues are physically destroyed by them.
  • shrinkage of tissues caused by evaporation of moisture at the time of freeze-drying causes destruction of tissues—in which bondage of collagen fibers composing most of the tissue is weakened or disconnected by destroying tissues due to ice crystals—to be accelerated.
  • the acellular dermal matrix prepared as such has weak tensile strength, so that it is difficult to expect good prognosis after surgery when it is transplanted at the joint sites of burn patients.
  • the maximum prevention of formation of ice crystals in tissues is the prerequisite of the cryoprotectant.
  • glycerol having a property of membrane permeability high concentration of sucrose and propylene glycol having a property of membrane impermeability are used as the major ingredients of cryopreservation solution to protect and stabilize tissues from physical destruction caused by ice crystals.
  • propylene glycol used in the present invention is used as a food additive and in cosmetics since it shows little toxicity compared with other glycols, and has antibiotic activity to prevent proliferation of bacteria and fungi.
  • the cryoprotectant of the present invention can protect tissues from contamination after freeze-drying.
  • the stability of tissue of the acellular dermal matrix prepared according to the present invention can be improved.
  • the optimal mixing ratio of sucrose, glycerol, propylene glycol, and the basic solvent or solution can further improve the stability of the dermal tissue.
  • the concentration of sucrose is less than 20% by weight, the stability of the tissue may be decreased due to ice crystals formed in a freezing step.
  • the concentration of sucrose is greater than 40% by weight, the stability of the tissue may be deteriorated by sugar crystals formed in the tissue after freeze-drying due to high concentration of sugar ingredients.
  • the cryoprotectant is most preferably prepared by dissolving sucrose in the basic constituents-mixed solution to the final concentration of 30% by weight.
  • the penetration of the cryoprotectant into the skin from which epidermis and cells in dermis are removed may be carried out according to conventional methods known in the art.
  • the cryoprotectant may be penetrated into the skin tissue in a low-temperature bath.
  • the sugar ingredient may be the cause of contamination as a nutrient source, it is preferable to treat a cryoprotectant at low temperature.
  • the treatment of cryoprotectant at room temperature may induce denaturation of collagen tissue which is a raw material, it is preferable to penetrate the cryoprotectant into the skin at low temperature rather than room temperature. Time needed for penetration may vary depending on the size of skin tissue and other factors.
  • the cryoprotectant may be penetrated into the skin tissue in a 4° C. low-temperature bath for about 6 to 24 hours.
  • the cryoprotectant-penetrated skin be frozen by using a freeze-dryer which can control the temperature rate.
  • a freeze-dryer that can control freezing rate allows the skin tissue to be frozen at a desired rate.
  • the freezing rate of skin with the freeze-dryer that can control freezing rate is preferably ⁇ 0.1° C. to ⁇ 5° C. per minute, and most preferably ⁇ 1° C. per minute.
  • the acellular dermal matrix for transplantation prepared according to the processing method of the present invention shows that the stability of the tissue is high, extracellular matrix and basement membrane are well maintained without impairment, and the change of biological properties is minimized. As a result, the success rate of acellular dermal matrix grafting can be increased, and treatment duration can be curtailed.
  • FIG. 1 is optical microscope photographs of acellular dermal matrix freeze-dried with a cryoprotectant using each concentration of sucrose after rehydration and H&E staining with 100 ⁇ magnification.
  • A 5% sucrose
  • B 10% sucrose
  • C 15% sucrose
  • D 20% sucrose
  • E 25% sucrose
  • F 30% sucrose
  • FIG. 2 is scanning electron microscope photographs of acellular dermal matrix freeze-dried with a cryoprotectant using each concentration of sucrose with 150 ⁇ and 1,000 ⁇ magnifications.
  • A 0% sucrose, 150 ⁇
  • B 0% sucrose, 1,000 ⁇
  • C 10% sucrose, 150 ⁇
  • D 10% sucrose, 1,000 ⁇
  • E 30% sucrose, 150 ⁇
  • F 30% sucrose, 1,000 ⁇
  • FIG. 3 is optical microscope photographs of acellular dermal matrixes of Example and Comparative Examples 1 and 2 with 100 ⁇ magnification.
  • A Example; B: Comparative Example 1; C: Comparative Example 2
  • FIG. 4 is a graph representing results of degradability measured by the treatment of acellular dermal matrix which is processed with cryoprotectants comprising sucrose in the final concentration of 30% by weight, and acellular dermal matrixes of Comparative Examples 1 and 2 with collagenase.
  • Resitive control treatment of collagen powder with collagenase
  • Negative control No treatment of collagenase
  • pig skin which is the closest to human skin—is used for preparing ten (10) of samples according to the following methods of Example and Comparative Examples.
  • An acellular dermal matrix was prepared with pig skin according to the following steps.
  • step (10) Sucrose (Sigma, USA) was added to the solution of step (10) as the final concentration of 30% by weight and dissolved to obtain a cryoprotectant.
  • a low-temperature bath (P-039, CoreTech, Korea) was set at 4° C.
  • step (9) The pig skin of step (9) was put in the 4° C. low-temperature bath, and then the cryoprotectant was penetrated into the pig skin for 12 hours.
  • a freezing dryer (Genesis 25XL, VirTis, USA) was prepared.
  • the Tyvek bag of step (14) was put in the freezing dryer and frozen to ⁇ 70° C. at the rate of ⁇ 1° C. per minute, and then dried under the vacuum of 5 torr for 24 hours to obtain a freeze-dried acellular dermal matrix.
  • freeze-dried acellular dermal matrix was sterilized in an E.O. gas sterilizer (HS-4313EO, HanShin Medical Co., Ltd., Korea).
  • An acellular dermal matrix was prepared with pig skin by using HBSS containing 7.5% dextran (MWT 70,000), 6% sucrose, 7.5% polyvinylpyrrolidone (MWT 40,000), 1.25% raffinose and 1 mM disodium ethylenediamine tetraacetic acid according to the same method disclosed in Example 1 of U.S. Pat. No. 5,336,616.
  • a freeze-dried skin was prepared with pig skin according to the following steps.
  • a low-temperature bath (P-039, CoreTech, Korea) was set at 4° C.
  • step (9) The pig skin of step (9) was put in the 4° C. low-temperature bath, and then the cryoprotectant was penetrated into the pig skin for 12 hours.
  • a freezing dryer (Genesis 25XL, VirTis, USA) was prepared.
  • the Tyvek bag of step (13) was put in the freezing dryer and frozen to ⁇ 70° C. at the rate of ⁇ 1° C. per minute, and then dried under the vacuum of 5 torr for 24 hours to obtain a freeze-dried acellular dermal matrix.
  • freeze-dried acellular dermal matrix was sterilized in an E.O. gas sterilizer (HS-4313EO, HanShin Medical Co., Ltd., Korea).
  • H&E (hematoxylin & eosin) staining was carried out as follows:
  • a paraffin block was cut with 4 ⁇ m thickness and dried to obtain a paraffin section.
  • a specimen was pre-fixed with 2.5% glutaraldehyde solution (fixative solution) for 2 hours, washed with 0.1M phosphate-buffered saline and post-fixed with 1% OsO 4 solution.
  • the freeze-dried acellular dermal matrixes were prepared according to the method of Example by using a cryoprotectant comprising sucrose in the final concentration of 5%, 10%, 15%, 20%, 25% and 30% by weight.
  • the prepared acellular dermal matrixes were rehydrated, and were then stained with H&E and photographed with an optical microscope (Olympus BX51). The results are represented in FIG. 1 .
  • the concentration of sucrose is less than 20% by weight, the matrix of tissue is destroyed in proportion to the concentration after freeze-drying.
  • sucrose concentration is 20%, 25% and 30% by weight, the matrix of tissue is not destroyed in proportion to the concentration of sucrose and maintains morphology close to the original structure.
  • the acellular dermal matrix prepared by using a cryoprotectant not containing sucrose and the acellular dermal matrixes prepared as above by treating with a cryoprotectant containing 10% and 30% by weight of sucrose were photographed with a scanning electron microscope according to the above method. The results are represented in FIG. 2 .
  • the acellular dermal matrixes are destroyed after freeze-drying.
  • the acellular dermal matrix treated with a cryoprotectant containing 30% by weight of sucrose shows that its morphology is well maintained after freeze-drying without destroying matrix of tissue.
  • Example and Comparative Examples 1 and 2 were stained with H&E and then photographed with an optical microscope (Olympus BX51) according to the above method. The results are represented in FIG. 3 .
  • the acellular dermal matrix of Example shows much better stability of tissue by preventing tissue from destruction in the course of freeze-drying as compared with the acellular dermal matrixes of Comparative Examples 1 and 2.
  • step (2) The sample of step (2) was treated with ninhydrin color reagent and absorbance at 570 nm was then measured.
  • the above calculated L-leucine release amount is represented in FIG. 4 .
  • the acellular dermal matrix of Example shows higher stability of tissue as compared with the acellular dermal matrixes of Comparative Examples 1 and 2 so that degradation rate by collagenase is remarkably reduced.
  • an acellular dermal matrix according to the present method can increase the success rate of grafting and curtail the treatment duration since its extracellular matrix and basement membrane are well maintained without impairment, and the change of biological properties is minimized.

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  • Health & Medical Sciences (AREA)
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KR1020100018134A KR101089614B1 (ko) 2010-02-26 2010-02-26 무세포 진피 기질의 제조 방법 및 그로부터 제조된 무세포 진피 기질
KR10-2010-0018134 2010-02-26
PCT/KR2010/003783 WO2011105663A1 (ko) 2010-02-26 2010-06-11 무세포 진피 기질의 제조 방법 및 그로부터 제조된 무세포 진피 기질

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